Structural basis for potassium transport in prokaryotes by KdpFABC.

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K+ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

Atomic Structure Optimization with Machine-Learning Enabled Interpolation between Chemical Elements.

We introduce a computational method for global optimization of structure and ordering in atomic systems. The method relies on interpolation between chemical elements, which is incorporated in a machine-learning structural fingerprint. The method is based on Bayesian optimization with Gaussian processes and is applied to the global optimization of Au-Cu bulk systems, Cu-Ni surfaces with CO adsorption, and Cu-Ni clusters. The method consistently identifies low-energy structures, which are likely to be the global minima of the energy. For the investigated systems with 23-66 atoms, the number of required energy and force calculations is in the range 3-75.

Impaired Renal HCO3- Excretion in Cystic Fibrosis.

BACKGROUND: Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis. METHODS: By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor. RESULTS: ß-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-. CONCLUSIONS: These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice.

The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the ß2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, ß2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.

Furosemide-induced urinary acidification is caused by pronounced H+ secretion in the thick ascending limb.

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 µM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 µM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.

Structural and functional characterization of human and murine C5a anaphylatoxins.

Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8(Δ71-73), and of murine C5a and C5a-desArg are reported. Whereas A8(Δ71-73) adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

Single-mode pumped high air-fill fraction photonic crystal fiber taper for high-power deep-blue supercontinuum sources.

Dispersion control with axially nonuniform photonic crystal fibers (PCFs) permits supercontinuum (SC) generation into the deep-blue from an ytterbium pump laser. In this Letter, we exploit the full degrees of freedom afforded by PCFs to fabricate a fiber with longitudinally increasing air-fill fraction and decreasing diameter directly on the draw-tower. We demonstrate SC generation extending down to 375 nm in one such monolithic fiber device that is single-mode at 1064 nm at the input end.

Association of adrenal steroids with metabolomic profiles in patients with primary and endocrine hypertension.

Introduction: Endocrine hypertension (EHT) due to pheochromocytoma/paraganglioma (PPGL), Cushing's syndrome (CS), or primary aldosteronism (PA) is linked to a variety of metabolic alterations and comorbidities. Accordingly, patients with EHT and primary hypertension (PHT) are characterized by distinct metabolic profiles. However, it remains unclear whether the metabolomic differences relate solely to the disease-defining hormonal parameters. Therefore, our objective was to study the association of disease defining hormonal excess and concomitant adrenal steroids with metabolomic alterations in patients with EHT. Methods: Retrospective European multicenter study of 263 patients (mean age 49 years, 50% females; 58 PHT, 69 PPGL, 37 CS, 99 PA) in whom targeted metabolomic and adrenal steroid profiling was available. The association of 13 adrenal steroids with differences in 79 metabolites between PPGL, CS, PA and PHT was examined after correction for age, sex, BMI, and presence of diabetes mellitus. Results: After adjustment for BMI and diabetes mellitus significant association between adrenal steroids and metabolites - 18 in PPGL, 15 in CS, and 23 in PA - were revealed. In PPGL, the majority of metabolite associations were linked to catecholamine excess, whereas in PA, only one metabolite was associated with aldosterone. In contrast, cortisone (16 metabolites), cortisol (6 metabolites), and DHEA (8 metabolites) had the highest number of associated metabolites in PA. In CS, 18-hydroxycortisol significantly influenced 5 metabolites, cortisol affected 4, and cortisone, 11-deoxycortisol, and DHEA each were linked to 3 metabolites. Discussions: Our study indicates cortisol, cortisone, and catecholamine excess are significantly associated with metabolomic variances in EHT versus PHT patients. Notably, catecholamine excess is key to PPGL's metabolomic changes, whereas in PA, other non-defining adrenal steroids mainly account for metabolomic differences. In CS, cortisol, alongside other non-defining adrenal hormones, contributes to these differences, suggesting that metabolic disorders and cardiovascular morbidity in these conditions could also be affected by various adrenal steroids.

The role of phase coherence in seeded supercontinuum generation.

The noise properties of a supercontinuum can be controlled by modulating the pump with a seed pulse. In this paper, we numerically investigate the influence of seeding with a partially phase coherent weak pulse or continuous wave. We demonstrate that the noise properties of the generated supercontinuum are highly sensitive to the degree of phase noise of the seed and that a nearly coherent seed pulse is needed to achieve a coherent pulse break-up and low noise supercontinuum. The specific maximum allowable linewidth of the seed laser is found to decrease with increasing pump power.

Transmyringeal ventilation tube insertion for unilateral Menière's disease: a protocol for a prospective, sham-controlled, double-blinded, randomized, clinical trial.

BACKGROUND: Menière's disease is an idiopathic disorder characterized by recurrent episodes of vertigo lasting more than 20 min, unilateral sensorineural hearing loss, and tinnitus. If vertigo attacks occur frequently, the patient is usually severely incapacitated. Currently, there is no consensus on the treatment of Menière's disease. The evidence regarding most treatment options is sparse due to a lack of randomized trials together with an often-spontaneous relief over time and a considerable placebo effect. Insertion of a transmyringeal tube is a simple and relatively safe, minimally invasive procedure and previous open-label trials have shown promising results. STUDY DESIGN: This is a prospective, sham-controlled, double-blinded, randomized, clinical trial. AIM: This trial aims to assess the effects of inserting a ventilation tube into the tympanic membrane compared with sham treatment for definite or probable unilateral Menière's disease according to the criteria formulated by the Classification Committee of the Bàràny Society. OUTCOMES: The primary outcome will be the number of spontaneous vertigo attacks lasting more than 20 min and time to treatment failure. In addition to the primary outcome, we will assess various secondary outcomes related to hearing, ear fullness, dizziness, and serious adverse events. SAMPLE SIZE: An estimated 104 participants in total or 52 participants in each group will be necessary. The primary analysis will be according to the intention-to-treat principle. The trial will be initiated in 2021 and is expected to end in 2025. TRIAL STATUS: ClinicalTrials.gov : NCT04835688 . Registered on April 8, 2021. PROTOCOL VERSION: 1.8, 26-09-2022. Date of first enrollment: October 1st, 2021. End of study: anticipated January 2025.

Dietary K+ acts as a genuine diuretic.

K+ balance in mammals relies on regulated renal K+ excretion matching unregulated fluctuating K+ intake. Upon a K+ rich meal, rapid and powerful K+ excretion is needed. Renal K+ secretion is stimulated by the increased tubular flow. We speculated that high K+ intake acutely increases urinary flow to stimulate K+ excretion. METHODS: Mice were K+ challenged through diets or gavage. Post K+ loading urinary output, osmolarity, [K+ ]u , [Na+ ]u , plasma osmolarity, [copeptin]p , [K+ ]p , and [Na+ ]p were measured. To locate the mechanism of K+ -induced diuresis in the glomerular/tubular system we measured creatinine excretion and assessed functional transport in isolated perfused TALs and CDs during an acute [K+ ]bl switch from 3.6 to 6.5 mM. Molecular adaptations of transport proteins involved in water reabsorption were investigated by immunoblotting. RESULTS: (1) Mice switched from a 1% to 2% K+ diet increased diuresis within 12 hours and reciprocally reduced diuresis when switched from 1% to 0.01% K+ diet. (2) A single K+ gavage load, corresponding to 25%-50% of daily K+ intake, induced 100% increase in diuresis within 30 minutes. This occurred despite augmented plasma osmolarity and AVP synthesis. (3) K+ gavage did not change GFR. (4) In isolated perfused TALs, shifting [K+ ]bl from 3.6 to 6.5 mM did not affect AVP-induced NaCl transport. (5) In sharp contrast, in isolated perfused CDs, shifting [K+ ]bl from 3.6 to 6.5 mM markedly reduced CD AVP sensitivity, ie inhibited water absorption. CONCLUSION: Dietary K+ loading induces a rapidly on-setting diuresis. The mechanism of K+ -induced diuresis involves desensitization of the CD to AVP.

Identification of glucocorticoid-related molecular signature by whole blood methylome analysis.

OBJECTIVE: Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. DESIGN: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. METHODS: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. RESULTS: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. CONCLUSIONS: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays.

Whole blood methylome-derived features to discriminate endocrine hypertension.

BACKGROUND: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. RESULTS: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods-Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine-predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. CONCLUSIONS: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder.

Predicting Hypertension Subtypes with Machine Learning Using Targeted Metabolites and Their Ratios.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

Identification of risk loci for primary aldosteronism in genome-wide association studies.

Primary aldosteronism affects up to 10% of hypertensive patients and is responsible for treatment resistance and increased cardiovascular risk. Here we perform a genome-wide association study in a discovery cohort of 562 cases and 950 controls and identify three main loci on chromosomes 1, 13 and X; associations on chromosome 1 and 13 are replicated in a second cohort and confirmed by a meta-analysis involving 1162 cases and 3296 controls. The association on chromosome 13 is specific to men and stronger in bilateral adrenal hyperplasia than aldosterone producing adenoma. Candidate genes located within the two loci, CASZ1 and RXFP2, are expressed in human and mouse adrenals in different cell clusters. Their overexpression in adrenocortical cells suppresses mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. Our study identifies the first risk loci for primary aldosteronism and highlights new mechanisms for the development of aldosterone excess.

Machine learning for classification of hypertension subtypes using multi-omics: A multi-centre, retrospective, data-driven study.

BACKGROUND: Arterial hypertension is a major cardiovascular risk factor. Identification of secondary hypertension in its various forms is key to preventing and targeting treatment of cardiovascular complications. Simplified diagnostic tests are urgently required to distinguish primary and secondary hypertension to address the current underdiagnosis of the latter. METHODS: This study uses Machine Learning (ML) to classify subtypes of endocrine hypertension (EHT) in a large cohort of hypertensive patients using multidimensional omics analysis of plasma and urine samples. We measured 409 multi-omics (MOmics) features including plasma miRNAs (PmiRNA: 173), plasma catechol O-methylated metabolites (PMetas: 4), plasma steroids (PSteroids: 16), urinary steroid metabolites (USteroids: 27), and plasma small metabolites (PSmallMB: 189) in primary hypertension (PHT) patients, EHT patients with either primary aldosteronism (PA), pheochromocytoma/functional paraganglioma (PPGL) or Cushing syndrome (CS) and normotensive volunteers (NV). Biomarker discovery involved selection of disease combination, outlier handling, feature reduction, 8 ML classifiers, class balancing and consideration of different age- and sex-based scenarios. Classifications were evaluated using balanced accuracy, sensitivity, specificity, AUC, F1, and Kappa score. FINDINGS: Complete clinical and biological datasets were generated from 307 subjects (PA=113, PPGL=88, CS=41 and PHT=112). The random forest classifier provided ∼92% balanced accuracy (∼11% improvement on the best mono-omics classifier), with 96% specificity and 0.95 AUC to distinguish one of the four conditions in multi-class ALL-ALL comparisons (PPGL vs PA vs CS vs PHT) on an unseen test set, using 57 MOmics features. For discrimination of EHT (PA + PPGL + CS) vs PHT, the simple logistic classifier achieved 0.96 AUC with 90% sensitivity, and ∼86% specificity, using 37 MOmics features. One PmiRNA (hsa-miR-15a-5p) and two PSmallMB (C9 and PC ae C38:1) features were found to be most discriminating for all disease combinations. Overall, the MOmics-based classifiers were able to provide better classification performance in comparison to mono-omics classifiers. INTERPRETATION: We have developed a ML pipeline to distinguish different EHT subtypes from PHT using multi-omics data. This innovative approach to stratification is an advancement towards the development of a diagnostic tool for EHT patients, significantly increasing testing throughput and accelerating administration of appropriate treatment. FUNDING: European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 633983, Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE (to Z.E. and F.B.), and Deutsche Forschungsgemeinschaft (CRC/Transregio 205/1).

The secretory KCa1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence.

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na(+) absorption and K(+) secretion. The electrogenic Na(+) absorption is mediated by epithelial Na(+) channel (ENaC) in surface cells. Previously, we identified the large conductance Ca(2+)-activated K(+) channel, K(Ca)1.1 or big potassium (BK) channel, as the only relevant K(+) secretory pathway in mouse distal colon. The exact localisation of K(Ca)1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K(Ca)1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na(+)/K(+)/2Cl(-) (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K(Ca)1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K(Ca)1.1 α-subunit were, however, under these circumstances substantially augmented (K(Ca)1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na(+) absorption and BK channel-mediated K(+) secretion are two independent processes. These findings show that K(Ca)1.1-mediated K(+) secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.

Python erythrocytes are resistant to α-hemolysin from Escherichia coli.

α-Hemolysin (HlyA) from Escherichia coli lyses mammalian erythrocytes by creating nonselective cation pores in the membrane. Pore insertion triggers ATP release and subsequent P2X receptor and pannexin channel activation. Blockage of either P2X receptors or pannexin channels reduces HlyA-induced hemolysis. We found that erythrocytes from Python regius and Python molurus are remarkably resistant to HlyA-induced hemolysis compared to human and Trachemys scripta erythrocytes. HlyA concentrations that induced maximal hemolysis of human erythrocytes did not affect python erythrocytes, but increasing the HlyA concentration 40-fold did induce hemolysis. Python erythrocytes were more resistant to osmotic stress than human erythrocytes, but osmotic stress tolerance per se did not confer HlyA resistance. Erythrocytes from T. scripta, which showed higher osmotic resistance than python erythrocytes, were as susceptible to HlyA as human erythrocytes. Therefore, we tested whether python erythrocytes lack the purinergic signalling known to amplify HlyA-induced hemolysis in human erythrocytes. P. regius erythrocytes increased intracellular Ca²âº concentration and reduced cell volume when exposed to 3 mM ATP, indicating the presence of a P2X7-like receptor. In addition, scavenging extracellular ATP or blocking P2 receptors or pannexin channels reduced the HlyA-induced hemolysis. We tested whether the low HlyA sensitivity resulted from low affinity of HlyA to the python erythrocyte membrane. We found comparable incorporation of HlyA into human and python erythrocyte membranes. Taken together, the remarkable HlyA resistance of python erythrocytes was not explained by increased osmotic resistance, lack of purinergic hemolysis amplification, or differences in HlyA affinity.

Change of cardiac function, but not form, in postprandial pythons.

Pythons are renowned for a rapid and pronounced postprandial growth of the heart that coincides with a several-fold elevation of cardiac output that lasts for several days. Here we investigate whether ventricular morphology is affected by digestive state in two species of pythons (Python regius and Python molurus) and we determine the cardiac right-to-left shunt during the postprandial period in P. regius. Both species experienced several-fold increases in metabolism and mass of the digestive organs by 24 and 48 h after ingestion of meals equivalent to 25% of body mass. Surprisingly there were no changes in ventricular mass or dimensions as we used a meal size and husbandry conditions similar to studies finding rapid and significant growth. Based on these data and literature we therefore suggest that postprandial cardiac growth should be regarded as a facultative rather than obligatory component of the renowned postprandial response. The cardiac right-to-left shunt, calculated on the basis of oxygen concentrations in the left and right atria and the dorsal aorta, was negligible in fasting P. regius, but increased to 10-15% during digestion. Such shunt levels are very low compared to other reptiles and does not support a recent proposal that shunts may facilitate digestion in reptiles.

Targeted Metabolomics as a Tool in Discriminating Endocrine From Primary Hypertension.

CONTEXT: Identification of patients with endocrine forms of hypertension (EHT) (primary hyperaldosteronism [PA], pheochromocytoma/paraganglioma [PPGL], and Cushing syndrome [CS]) provides the basis to implement individualized therapeutic strategies. Targeted metabolomics (TM) have revealed promising results in profiling cardiovascular diseases and endocrine conditions associated with hypertension. OBJECTIVE: Use TM to identify distinct metabolic patterns between primary hypertension (PHT) and EHT and test its discriminating ability. METHODS: Retrospective analyses of PHT and EHT patients from a European multicenter study (ENSAT-HT). TM was performed on stored blood samples using liquid chromatography mass spectrometry. To identify discriminating metabolites a "classical approach" (CA) (performing a series of univariate and multivariate analyses) and a "machine learning approach" (MLA) (using random forest) were used.The study included 282 adult patients (52% female; mean age 49 years) with proven PHT (n = 59) and EHT (n = 223 with 40 CS, 107 PA, and 76 PPGL), respectively. RESULTS: From 155 metabolites eligible for statistical analyses, 31 were identified discriminating between PHT and EHT using the CA and 27 using the MLA, of which 16 metabolites (C9, C16, C16:1, C18:1, C18:2, arginine, aspartate, glutamate, ornithine, spermidine, lysoPCaC16:0, lysoPCaC20:4, lysoPCaC24:0, PCaeC42:0, SM C18:1, SM C20:2) were found by both approaches. The receiver operating characteristic curve built on the top 15 metabolites from the CA provided an area under the curve (AUC) of 0.86, which was similar to the performance of the 15 metabolites from MLA (AUC 0.83). CONCLUSION: TM identifies distinct metabolic pattern between PHT and EHT providing promising discriminating performance.